1. Replication fork stalling in late S-phase elicits nascent strand degradation by DNA mismatch repair.
- Author
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Colicino-Murbach E, Hathaway C, and Dungrawala H
- Subjects
- Humans, DNA metabolism, DNA genetics, BRCA2 Protein metabolism, BRCA2 Protein genetics, MRE11 Homologue Protein metabolism, MRE11 Homologue Protein genetics, DNA Repair Enzymes metabolism, DNA Repair Enzymes genetics, Heterochromatin metabolism, Heterochromatin genetics, Chromatin metabolism, DNA Mismatch Repair, S Phase genetics, DNA Replication, Exodeoxyribonucleases metabolism, Exodeoxyribonucleases genetics, Rad51 Recombinase metabolism, Rad51 Recombinase genetics
- Abstract
Eukaryotic chromosomal replication occurs in a segmented, temporal manner wherein open euchromatin and compact heterochromatin replicate during early and late S-phase respectively. Using single molecule DNA fiber analyses coupled with cell synchronization, we find that newly synthesized strands remain stable at perturbed forks in early S-phase. Unexpectedly, stalled forks are susceptible to nucleolytic digestion during late replication resulting in defective fork restart. This inherent vulnerability to nascent strand degradation is dependent on fork reversal enzymes and resection nucleases MRE11, DNA2 and EXO1. Inducing chromatin compaction elicits digestion of nascent DNA in response to fork stalling due to reduced association of RAD51 with nascent DNA. Furthermore, RAD51 occupancy at stalled forks in late S-phase is diminished indicating that densely packed chromatin limits RAD51 accessibility to mediate replication fork protection. Genetic analyses reveal that susceptibility of late replicating forks to nascent DNA digestion is dependent on EXO1 via DNA mismatch repair (MMR) and that the BRCA2-mediated replication fork protection blocks MMR from degrading nascent DNA. Overall, our findings illustrate differential regulation of fork protection between early and late replication and demonstrate nascent strand degradation as a critical determinant of heterochromatin instability in response to replication stress., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2024
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